Treatment of degenerative joint disease

ABSTRACT

The invention provides a method of treating a degenerative joint disease. The method comprises administering an effective amount of a pharmaceutical composition comprising a diketopiperazine with amino acid side chains of aspartic acid and alanine (DA-DKP). The invention also provides a pharmaceutical product as well as a kit comprising DA-DKP.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/453,573, filed Mar. 8, 2017, which is a continuation of U.S.application Ser. No. 14/604,479, filed Jan. 23, 2015, now U.S. Pat. No.9,623,072, which is a continuation of U.S. application Ser. No.14/350,634, filed Apr. 9, 2014, now U.S. Pat. No. 8,980,834, which is anational stage application under 35 U.S.C. 371 and claims the benefit ofPCT Application No. PCT/US2012/059455 having an international filingdate of Oct. 10, 2012, which designated the United States, which PCTapplication claims the benefit of priority under 35 U.S.C. § 119(e) toU.S. Provisional Patent Application No. 61/545,474, filed Oct. 10, 2011and U.S. Provisional Patent Application No. 61/561,221, filed Nov. 17,2011, the entire disclosures of each are incorporated herein byreference.

FIELD OF INVENTION

The invention relates to a method of treating a degenerative jointdisease. The method comprises administering an effective amount of apharmaceutical composition comprising a diketopiperazine with amino acidside chains of aspartic acid and alanine (DA-DKP). The invention alsoprovides a pharmaceutical product comprising DA-DKP.

BACKGROUND

Osteoarthritis is the most common form of arthritis, affecting 25 to 35million people in the U.S. Chronic pain and disability of osteoarthritisis initially caused by inflammatory responses in joint cartilage andbone that gradually worsens over time. Symptomatic osteoarthritis of theknee occurs in 10 to 13% of persons aged 60 and over. Kneeosteoarthritis alone increases the risk of loss of mobility, such asneeding assistance walking or climbing stairs, greater than for anyother medical condition in people aged 65 and over.

Current drug treatment for osteoarthritis of the knee is limited toanalgesics, non-steroidal anti-inflammatory drugs (NSAIDs) andintra-articular steroid injections, all of which have significantlimitations due to adverse effects. Despite these medical treatments,chronic knee osteoarthritis often causes progressive disabilityrequiring total joint replacement. The increasing prevalence ofosteoarthritis of the knee due to aging and obese populations suggests agrowing clinical need for safe and effective local knee treatments thatwill delay and potentially eliminate the need for more extensivesurgical treatments.

SUMMARY OF INVENTION

One embodiment of the invention relates to a method of treating adegenerative joint disease by administering to an animal in need thereofan effective amount of a pharmaceutical composition comprising DA-DKP.In one aspect, the degenerative joint disease is osteoarthritis.

In another aspect, the composition is administered no more frequentlythan once every six months, once every 5 months, once every 4 months,once every 3 months, or once every 2 months.

In yet another aspect, the composition can be administered by variousadministration routes. For example, the administration route may be bylocal administration, topical administration, or injection. In oneaspect, administration by injection is by intra-articular injection. Inyet another aspect, the composition administered by intra-articularinjection is a composition having a concentration of DA-DKP from about50 μM to about 350 μM.

In still another aspect, the composition further includesN-acetyl-tryptophan (NAT), caprylic acid, caprylate or combinationsthereof. In yet another aspect, the concentration of NAT, caprylic acid,caprylate or combinations thereof in the composition may be about 4 mMto about 20 mM.

In yet other aspects of the method, the DA-DKP is in a compositionprepared by removing albumin from a solution of a human serum albumincomposition. For example, the step of removing can be by treating thehuman serum albumin composition by a separation method. Such separationmethods can include ultrafiltration, sucrose gradient centrifugation,chromatography, salt precipitation, and sonication. In addition, thestep of removing can be by passing the human serum albumin compositionover an ultrafiltration membrane with a molecular weight cut off thatretains the albumin, and the resulting filtrate contains the DA-DKP. Inone aspect, the ultrafiltration membrane has a molecular weight cutoffof less than 50 kDa. In still another aspect, the ultrafiltrationmembrane has a molecular weight cut off less than 40 kDa, less than 30kDa, less than 20 kDa, less than 10 kDa, less than 5 kDa or less than 3kDa. In still another aspect, this composition further comprises NAT,caprylic acid, caprylate or combinations thereof. In yet another aspect,the concentration of NAT, caprylic acid, caprylate or combinationsthereof in the composition may be about 4 mM to about 20 mM.

In another aspect, the method of the invention can further includeadministering a second drug. For example, the second drug can be ananalgesic, an anti-inflammatory drug, or combinations thereof.

Another embodiment of the invention is a pharmaceutical productcomprising a DA-DKP-containing composition formulated for administrationby injection. In one aspect, the product is formulated foradministration by intra-articular injection. In another aspect theDA-DKP is prepared by removing albumin from a solution of a human serumalbumin composition. In one aspect, the step of removing the albumin canbe by treating the human serum albumin composition by a separationmethod. For example, the separation method can be ultrafiltration,sucrose gradient centrifugation, chromatography, salt precipitation, orsonication. In addition, the step of removing can be by passing thehuman serum albumin composition over an ultrafiltration membrane with amolecular weight cut off that retains the albumin, and the resultingfiltrate contains DA-DKP. In one aspect, the ultrafiltration membranehas a molecular weight cutoff of less than 50 kDa. In still anotheraspect, the ultrafiltration membrane has a molecular weight cut off lessthan 40 kDa, less than 30 kDa, less than 20 kDa, less than 10 kDa, lessthan 5 kDa or less than 3 kDa.

In yet another aspect, the DA-DKP of the pharmaceutical product that isadministered by intra-articular injection is a composition having aconcentration of DA-DKP from about 50 μM to about 350 μM.

In still another aspect, the product contains the DA-DKP compositionthat further contains NAT, caprylic acid, caprylate or combinationsthereof. In yet another aspect, the concentration of NAT, caprylic acid,caprylate or combinations thereof in the product may be about 4 mM toabout 20 mM.

In yet another aspect, the pharmaceutical product, further contains apharmaceutically-acceptable carrier.

Another embodiment of the invention relates to a kit that includes apharmaceutical product. In one aspect the product includes aDA-DKP-containing composition formulated for administration byinjection. In still another aspect, the DA-DKP in the kit is prepared byremoving albumin from a solution of a human serum albumin composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the mean change in pain numerical rating scale (NRS) forsubjects treated with a single 10 ml injection in one knee of Ampion™(<5000 MW fraction) combined with a steroid (betamethasone)/lidocainesuspension or injected with a saline placebo combined with steroid(betamethasone)/lidocaine suspension. The scores were completed at 6hours post-dose on Day 1, 24 hours post-dose on Day 2 and 72 hourspost-dose on Day 4.

FIG. 2 shows the mean change in pain NRS as described in FIG. 1 andincludes a repeated measures analysis of variance (ANOVA).

FIG. 3A shows the absolute difference in WOMAC (Western Ontario McMasterUniversity Osteoarthritis Index 3.1) pain subscores at 24 hours for thetreatment described in FIG. 1.

FIG. 3B shows the absolute difference in WOMAC pain subscores at 72hours for the treatment described in FIG. 1.

FIG. 4A shows the absolute difference in WOMAC stiffness subscores at 24hours for the treatment described in FIG. 1.

FIG. 4B shows the absolute difference in WOMAC stiffness subscores at 72hours for the treatment described in FIG. 1.

FIG. 5A shows the absolute difference in WOMAC function subscores at 24hours for the treatment described in FIG. 1.

FIG. 5B shows the absolute difference in WOMAC function subscores at 72hours for the treatment described in FIG. 1.

FIG. 6 shows a potential steroid time course.

FIG. 7 shows the mean change from baseline across all subject visits forthe treatment described in FIG. 1. (“LS” stands for least squares)

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of treating a degenerative jointdisease. The treatment comprises administering an effective amount of apharmaceutical composition comprising aspartyl-alanyl diketopiperazine(DA-DKP) to an individual having a need thereof. DA-DKP has multipleanti-inflammatory and immune modulating effects including inhibition ofmultiple pro-inflammatory cytokines, chemokines and signaling moleculesat the transcription level, inhibition of the migration and adhesion ofT-cells and monocytes, activity at the G-coupled protein receptor level,activity on actin-dependent cytoskeletal events, reduction in vascularpermeability and inhibition of inflammation induced by plateletactivating factor. As described in more detail below, the effects ofDA-DKP on degenerative joint disease have been found to be unexpectedlylong lasting and in some studies were found to increase in time ascompared to the use of steroids.

The invention also provides for a pharmaceutical product comprising aDA-DKP composition. The DA-DKP of the product may be prepared byremoving albumin from a solution of human serum albumin.

The invention also provides for kit comprising a DA-DKP compositionformulated for administration by injection.

A degenerative joint disease is a gradual deterioration of the articularcartilage that covers joints. A degenerative joint disease(osteoarthritis) is a noninfectious progressive disorder of theweightbearing joints. The normal articular joint cartilage is smooth,white, and translucent. It is composed of cartilage cells (chondrocytes)imbedded in a sponge-like matrix made of collagen, proteinpolysaccharides, and water. With early primary arthritis, the cartilagebecomes yellow and opaque with localized areas of softening androughening of the surfaces. As degeneration progresses, the soft areasbecome cracked and worn, exposing bone under the cartilage. The bonethen begins to remodel and increase in density while any remainingcartilage begins to fray. Eventually, osteophytes (spurs of new bone)covered by cartilage form at the edge of the joint. As mechanical wearincreases, the cartilage needs repairing. The cartilage cells are unableto produce enough of the sponge-like matrix and therefore the damagedcartilage cannot repair itself. The cartilage has no blood supply toenhance healing. The majority of degenerative joint disease is theresult of mechanical instabilities or aging changes within the joint.This includes old age degenerative arthritis and, in youngerindividuals, may be the result of injuries, bruises, abnormal jointconfiguration (i.e. hip dysplasia), or mechanical wear from anteriorcruciate ligament rupture, patellar luxation, or osteochondritisdissecans, for example. Degenerative joint disease can occur at anyjoint in the body, including without limitation, knee, hip, shoulder,hand and spine.

Conventional pharmaceutical therapies for degenerative joint diseaseinclude acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDS),narcotics, and corticosteroids.

“Treat” is used herein to mean to reduce (wholly or partially) thesymptoms, duration or severity of a disease.

The pharmaceutical composition comprising DA-DKP of the invention isadministered to an animal in need of treatment. Preferably, the animalis a mammal, such as a rabbit, goat, dog, cat, horse or human. Effectivedosage amounts may vary with the severity of the disease or condition,the route(s) of administration, the duration of the treatment, theidentify of any other drugs being administered to the animal, the age,size and species of the animal, and like factors known in the medicaland veterinary arts.

Because the treatment of the present invention provides a long-lastingeffect on the symptoms of degenerative joint disease, one aspect of thepresent invention is that the composition comprising DA-DKP may beadministered to an animal at longer time intervals than would beexpected for conventional therapies. For example, the presentcomposition can be administered no more frequently than once every sixmonth, once every five months, once every four months, once every threemonths, once every two months, once every month, once every four weeks,once every three weeks, once every two weeks or once every week.

The composition of the present invention comprising DA-DKP may beadministered to an animal patient for therapy by any suitable route ofadministration, including locally, parenterally (e.g., injection,intra-articular injection, intravenously, intraspinally,intraperitoneally, subcutaneously, or intramuscularly), transdermally,and topically. A preferred route of administration is intra-articularinjection.

The composition of the present invention may be a pharmaceuticalsolution having a DA-DKP concentration range with a lower endpoint ofabout 10 about 20 about 30 μM, about 40 μM, about 50 μM, about 60 μM,about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 110 μM, about120 μM, about 130 μM, about 140 μM, about 150 μM, about 160 μM, about170 μM, about 180 μM, about 190 μM, about 200 μM, about 210 μM, about220 μM, about 230 μM, about 240 μM, about 240, about 250 μM, about 260μM, about 270 μM, about 280 μM, about 290 μM, about 300 μM, about 310,about 320 μM, about 330 μM, about 340 μM, about 350 μM, about 360 μM,about 370 μM, about 380 μM, about 390 μM, or about 400 μM. Thecomposition of the present invention may be a pharmaceutical solutionhaving a DA-DKP concentration range with an upper endpoint of about 600μM, about 580 μM, about 570 μM, about 560 μM, about 550 μM, about 540μM, about 530 μM, about 520 μM, about 510 μM, about 500 μM, about 490μM, about 480 μM, about 470 μM, about 460 μM, about 450 μM, about 440μM, about 430 μM, about 420 μM, about 410 μM, about 400 μM, about 390μM, about 380 μM, about 370 μM, about 360 μM, about 350, about 340 μM,about 330 μM, about 320 μM, about 310 μM, about 300 μM, about 290 μM,about 280, about 270 μM, about 260 μM, about 250 μM, about 240 μM, about230 μM, about 220 μM, about 210 μM, or about 200 μM.

An effective amount of DA-DKP in the composition of the presentinvention for treating a degenerative joint disease or condition can bea range with a lower endpoint of about 10 μg, about 15 μg, about 20 μg,about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg,about 80 μg, about 85 μg, about 90 μg, about 95 μg, about 100 μg, about110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about160 μg, about 170 μg, about 180 μg, about 190 μg, about 200 μg, about210 μg, about 220 μg, about 230 μg, about 240 μg, about 250 μg, about260 μg, about 270 μg, about 280 μg, about 290 μg, about 300 μg, about310 μg, about 320 μg, about 330 μg, about 340 μg, about 350 μg, about360 μg, about 370 μg, about 380 μg, about 390 μg, about 400 μg, about425 μg, about 450 μg, about 475 μg or about 500 μg. In addition, aneffective amount of DA-DKP in the composition of the present inventionfor treating a degenerative joint disease or condition can be a rangewith upper endpoint of about 500 μg, about 490 μg, about 480 μg, about470 μg, about 460 μg, about 450 μg, about 440 μg, about 430 μg, about420 μg, about 410 μg, about 400 μg, about 390 μg, about 380 μg, about370 μg, about 360 μg, about 350 μg, about 340 μg, about 330 μg, about320 μg, about 310 μg, about 300 μg, about 290 μg, about 280 μg, about270 μg, about 260 μg, about 250 μg, about 240 μg, about 230 μg, about220 μg, about 210 μg, about 200 μg, about 190 μg, about 180 μg, about170 μg, about 160 μg, about 150 μg, about 140 μg, about 130 μg, about120 μg, about 110 μg, about 100 μg, about 90 μg, about 80 μg, about 70μg, about 60 μg, about 50 μg, about 40 μg, about 30 μg, or about 20 μg.

Dosage forms for the topical or transdermal administration of compoundsof the invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches, and drops. The active ingredient maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to theactive ingredient, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to the active ingredient,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder or mixtures of these substances.Sprays can additionally contain customary propellants such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of compounds of the invention to the body. Such dosage formscan be made by dissolving, dispersing or otherwise incorporating one ormore compounds of the invention in a proper medium, such as anelastomeric matrix material. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate of such fluxcan be controlled by either providing a rate-controlling membrane ordispersing the compound in a polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteraladministrations comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or non-aqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, solutes which render the formulation isotonicwith the blood of the intended recipient or suspending or thickeningagents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as wetting agents,emulsifying agents and dispersing agents. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the likein the compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monosterate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drug isaccomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer, and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue. The injectable materials can be sterilized forexample, by filtration through a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampoules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

Kits comprising the pharmaceutical products of the present invention arealso provided. The kits can comprise a DA-DKP composition formulated foradministration by injection. The DA-DKP can be prepared as describedherein, such as by removing albumin from a solution of a human albumincomposition. The kits may contain unit-dose or multi-dose sealedcontainers, for example, ampoules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use. Thekits may also be stored in a condition, wherein the contents are readyfor direct use or injection.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition). The pharmaceuticalcompositions of the invention comprise a compound or compounds of theinvention as an active ingredient in admixture with one or morepharmaceutically-acceptable carriers and, optionally, with one or moreother compounds, drugs or other materials. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the animal.Pharmaceutically-acceptable carriers are well known in the art.Regardless of the route of administration selected, the compounds of thepresent invention are formulated into pharmaceutically-acceptable dosageforms by conventional methods known to those of skill in the art. See,e.g., Remington's Pharmaceutical Sciences.

The composition of the present invention may further compriseN-acetyl-tryptophan (NAT), caprylic acid, caprylate or combinationsthereof. Preferably, the composition may comprise NAT. Compositions ofthe present invention having NAT, caprylic acid, caprylate orcombinations thereof may be a pharmaceutical composition having a NAT,caprylic acid, caprylate or combinations thereof concentration rangewith a lower endpoint of about 1 mM, about 2 mM, about 3 mM, about 4 mM,about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM,about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM. Inaddition, compositions of the present invention having NAT, caprylicacid, caprylate or combinations thereof may be a pharmaceuticalcomposition having a NAT, caprylic acid, caprylate or combinationsthereof concentration range with an upper endpoint of about 40 mM, about39 mM, about 38 mM, about 37 mM, about 36 mM, about 35 mM, about 34 mM,about 33 mM, about 32 mM, about 31 mM, about 30 mM, about 29 mM, about28 mM, about 27 mM, about 26 mM, about 25 mM, about 24 mM, about 23 mM,about 22, or about 21 mM. Preferably, the concentration range is about 4mM to about 20 mM.

In addition, the composition of the present invention may also comprisea second drug such as an analgesic (such as lidocaine or paracetoamol),an anti-inflammatory (such as bethamethasone, non-steroidanti-inflammatory drugs (NSAIDs), acetaminophen, ibuprofen, naproxen),and/or other suitable drugs.

Methods of making diketopiperazines, such as DA-DKP, are well known inthe art, and these methods may be employed to synthesize thediketopiperazines of the invention. See, e.g., U.S. Pat. Nos. 4,694,081,5,817,751, 5,990,112, 5,932,579 and 6,555,543, US Patent ApplicationPublication Number 2004/0024180, PCT applications WO 96/00391 and WO97/48685, and Smith et al., Bioorg. Med. Chem. Letters, 8, 2369-2374(1998), the complete disclosures of which are incorporated herein byreference.

For instance, diketopiperazines, such as DA-DKP, can be prepared byfirst synthesizing dipeptides. The dipeptides can be synthesized bymethods well known in the art using L-amino acids, D-amino acids or acombination of D- and L-amino acids. Preferred are solid-phase peptidesynthetic methods. Of course, dipeptides are also available commerciallyfrom numerous sources, including DMI Synthesis Ltd., Cardiff, UK (customsynthesis), Sigma-Aldrich, St. Louis, Mo. (primarily custom synthesis),Phoenix Pharmaceuticals, Inc., Belmont, Calif. (custom synthesis),Fisher Scientific (custom synthesis) and Advanced ChemTech, Louisville,Ky.

Once the dipeptide is synthesized or purchased, it is cyclized to form adiketopiperazine. This can be accomplished by a variety of techniques.For example, U.S. Patent Application Publication Number 2004/0024180describes a method of cyclizing dipeptides. Briefly, the dipeptide isheated in an organic solvent while removing water by distillation.Preferably, the organic solvent is a low-boiling azeotrope with water,such as acetonitrile, allyl alcohol, benzene, benzyl alcohol, n-butanol,2-butanol, t-butanol, acetic acid butylester, carbon tetrachloride,chlorobenzene chloroform, cyclohexane, 1,2-dichlorethane, diethylacetal,dimethylacetal, acetic acid ethylester, heptane, methylisobutylketone,3-pentanol, toluene and xylene. The temperature depends on the reactionspeed at which the cyclization takes place and on the type ofazeotroping agent used. The reaction is preferably carried out at50-200° C., more preferably 80-150° C. The pH range in which cyclizationtakes place can be easily determine by the person skilled in the art. Itwill advantageously be 2-9, preferably 3-7.

When one or both of the amino acids of the dipeptide has, or isderivatized to have, a carboxyl group on its side chain (e.g., asparticacid or glutamic acid), the dipeptide is preferably cyclized asdescribed in U.S. Pat. No. 6,555,543. Briefly, the dipeptide, with theside-chain carboxyl still protected, is heated under neutral conditions.Typically, the dipeptide will be heated at from about 80° C. to about180° C., preferably at about 120° C. The solvent will be a neutralsolvent. For instance, the solvent may comprise an alcohol (such asbutanol, methanol, ethanol, and higher alcohols, but not phenol) and anazeotropic co-solvent (such as toluene, benzene, or xylene). Preferably,the alcohol is butan-2-ol, and the azeotropic co-solvent is toluene. Theheating is continued until the reaction is complete, and such times canbe determined empirically. Typically, the dipeptide will be cyclized byrefluxing it for about 8-24 hours, preferably about 18 hours. Finally,the protecting group is removed from the diketopiperazine. In doing so,the use of strong acids (mineral acids, such as sulfuric or hydrochloricacids), strong bases (alkaline bases, such as potassium hydroxide orsodium hydroxide), and strong reducing agents (e.g., lithium aluminumhydride) should be avoided, in order to maintain the chirality of thefinal compound.

Dipeptides made on solid phase resins can be cyclized and released fromthe resin in one step. See, e.g., U.S. Pat. No. 5,817,751. For instance,the resin having an N-alkylated dipeptide attached is suspended intoluene or toluene/ethanol in the presence of acetic acid (e.g., 1%) ortriethylamine (e.g., 4%). Typically, basic cyclization conditions arepreferred for their faster cyclization times.

Other methods of cyclizing dipeptides and of making diketopiperazinesare known in the art and can be used in the preparation ofdiketopiperazines useful in the practice of the invention. See, e.g.,those references listed above. In addition, many diketopiperazinessuitable for use in the present invention can be made as described belowfrom proteins and peptides. Further, diketopiperazines for use in thepractice of the invention can be obtained commercially from, e.g., DMISynthesis Ltd., Cardiff, UK (custom synthesis).

The DA-DKP composition and/or products of the present invention can beprepared from solutions containing DA-DKP, including from thecommercially-available pharmaceutical compositions comprising albumin,such as human serum albumin, by well known methods, such asultrafiltration, chromatography (size-exclusion chromatography (e.g.,Centricon filtration), affinity chromatography (e.g., using a column ofbeads having attached thereto an antibody or antibodies directed to thedesired diketopiperazine(s) or an antibody or antibodies directed to thetruncated protein or peptide), anion exchange or cation exchange),sucrose gradient centrifugation, chromatography, salt precipitation, orsonication, that will remove some or all of the albumin in the solution.The resultant DA-DKP-containing composition and/or product can be usedand incorporated into pharmaceutical compositions as described above.

Using an ultrafilration separation method, a human serum albumincomposition can be passed over an ultrafiltration membrane having amolecular weight cut-off that retains the albumin while the DA-DKPpasses into the resulting filtrate or fraction. This filtrate maycomprise components having molecular weights less than about 50 kDA,less than about 40 kDa, less than 30 kDa, less than about 20 kDa, lessthan about 10 kDa, less than about 5 kDa, less than about 3 kDa.Preferably, the filtrate comprises components having molecular weightsless than about 5 Da (also referred to as “<5000 MW”). This <5000 MWfraction or filtrate contains DA-DKP which is formed after the dipeptideaspartate-alanine is cleaved from albumin and subsequently cyclized intothe diketopiperazine.

Physiologically-acceptable salts of the DA-DKP of the invention may alsobe used in the practice of the invention. Physiologically-acceptablesalts include conventional non-toxic salts, such as salts derived frominorganic acids (such as hydrochloric, hydrobromic, sulfuric,phosphoric, nitric, and the like), organic acids (such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,glutamic, aspartic, benzoic, salicylic, oxalic, ascorbic acid, and thelike) or bases (such as the hydroxide, carbonate or bicarbonate of apharmaceutically-acceptable metal cation or organic cations derived fromN,N-dibenzylethylenediamine, D-glucosamine, or ethylenediamine). Thesalts are prepared in a conventional manner, e.g., by neutralizing thefree base form of the compound with an acid.

As used herein, “a” or “an” means one or more.

As used herein, “comprises” and “comprising” include within their scopeall narrower terms, such as “consisting essentially of” and “consistingof” as alternative embodiments of the present invention characterizedherein by “comprises” or “comprising”. In regard to use of “consistingessentially of”, this phrase limits the scope of a claim to thespecified steps and materials and those that do not materially affectthe basic and novel characteristics of the invention disclosed herein.

Additional objects, advantages and novel features of the presentinvention will become apparent to those skilled in the art byconsideration of the following non-limiting examples. The followingexperimental results are provided for purposes of illustration and arenot intended to limit the scope of the invention.

EXAMPLES Example 1

A clinical trial was performed to investigate the effect ofintra-articular knee injection of a <5000 MW fraction of human serumalbumin (also referred to herein as “Ampion™”) for improving jointfunction and reducing the pain of osteoarthritis of the knee. Arandomized, placebo-controlled, double-blind, parallel study with 60evaluable subjects was chosen as the appropriate design to estimate thetreatment effect and safety of the <5000 MW Fraction when it wasinjected into the study knee.

Primary Objective:

To investigate the reduction of pain in subjects with kneeosteoarthritis of a single 10 ml intra-articular injection containingthe <5000 MW Fraction combined with lidocaine/betamethasone suspensioncompared with the <5000 MW Fraction combined with betamethasonesuspension or compared with a saline placebo combined withlidocaine/betamethasone suspension.

Secondary Objectives:

To investigate joint function and pain in subjects with kneeosteoarthritis, as assessed by the Western Ontario McMaster UniversityOsteoarthritis (WOMAC) Index (Bellamy et al., “Validation study ofWOMAC: a health status instrument for measuring clinically importantpatient relevant outcomes to antirheumatic drug therapy in patients withosteoarthritis of the hip or knee.” J Rheumatology 1988; 15:1833-1840)of a single intra-articular injection of the <5000 MW Fraction combinedwith lidocaine/betamethasone suspension compared with the <5000 MWFraction combined with betamethasone suspension or compared with asaline placebo combined with lidocaine/betamethasone suspension.

To investigate the requirement for rescue medications in subjects withknee osteoarthritis, after a single intra-articular injection of the<5000 MW Fraction combined with lidocaine/betamethasone suspensioncompared with the <5000 MW Fraction combined with betamethasonesuspension or compared with a saline placebo combined withlidocaine/betamethasone suspension.

To investigate the effect on the range of motion in subjects with kneeosteoarthritis and limited range of motion in the study knee due to painand inflammation, after a single intra-articular injection of the <5000MW Fraction combined with lidocaine/betamethasone suspension comparedwith the <5000 MW Fraction combined with betamethasone suspension orcompared with a saline placebo combined with lidocaine/betamethasonesuspension.

To compare safety and tolerability between treatments as assessed byreported adverse events in the study population.

Description of the Protocol

This study was a randomized, placebo-controlled, double-blind, parallelstudy designed to evaluate the effect of intra-articular knee injectionof the <5000 MW Fraction in male or female subjects ≥40 years old withsymptomatic primary knee osteoarthritis for 6 months precedingscreening. The study consisted of a 3 week screening period and a 4 daystudy participation period. Each subject was randomized to receive asingle 10 mL knee injection of one of the following:

-   -   the <5000 MW Fraction combined with lidocaine/betamethasone        suspension    -   the <5000 MW Fraction combined with betamethasone suspension        saline placebo combined with lidocaine/betamethasone suspension

Subjects were allowed to leave the clinic following a satisfactorypost-dose review by the investigator. Follow-up assessments wereperformed at 6 hours, 24 hours (Day 2) and 72 hours (Day 4)post-injection. These assessments were conducted at the clinic orexternally (e.g. home visit), at the discretion of the investigator.

Duration of Study Participation:

Screening: Between Day −21 and Day −1.

Treatment Period: A single injection in the study knee with clinicalfollow-up over 72 hours, including dosing on Day 1 and follow-up visitson Day 2 and Day 4.

The total duration of study participation was approximately 72 hours foreach subject, with a screening interval of up to 21 days.

Study Treatments

Each subject received a single 10 mL injection in one knee of one of thefollowing treatments:

-   -   <5000 MW Fraction combined with lidocaine/betamethasone        suspension    -   <5000 MW Fraction combined with betamethasone suspension    -   saline placebo combined with lidocaine/betamethasone suspension

Study Population

The study population was 60 patients, male or female, 40-85 years old,fully ambulatory, with symptomatic primary knee osteoarthritis for morethan 6 months prior to screening with Kellgren Lawrence Grade II or III.

Description of Investigational Product

Sterile 2 mL<5000 MW Fraction in rubber stopper storage vials wasprepared by Sypharma Pty Ltd., Dandenong, Victoria, Australia. The <5000MW Fraction was combined with either lidocaine/betamethasone suspensionor betamethasone suspension in a blinded fashion prior to injection.Saline placebo combined with lidocaine/betamethasone suspension wasadministered as a third treatment. See Table 1

TABLE 1 Investigational Product Components: <5000 MW FractionBethamethasone Lidocaine Saline Treatment A 4 mL 2 mL 4 mL Nil TreatmentB 4 mL 2 mL Nil 4 mL Treatment C Nil 2 mL 4 mL 4 mL

A single 10 mL injection of study drug combined withlidocaine/betamethasone suspension or combined with betamethasonesuspension was injected into the knee joint space under sterile prepconditions using an 18-21 gauge needle.

Study drug was stored at room temperature (59°-77° F. or 15°-25° C.) ina secure area with restricted access.

Randomization and Allocation to Study Treatment

Subjects were allocated to a sequentially numbered treatment inaccordance with the randomization schedule following confirmation ofeligibility at pre-dose. Each subject was assigned to one of the threetreatments, i.e. active <5000 MW Fraction (either combined withlidocaine/betamethasone or betamethasone suspension) or saline placebocombined with lidocaine/betamethasone. The allocation of treatment wasperformed using a block randomization algorithm.

If both knees were osteoarthritic, then at Screening the investigatorselected one knee to be the study knee, being the knee that bestsatisfied the requirements for the study. At the time of doseadministration, the study knee received investigational product inaccordance with the randomization schedule. The other knee receivednormal standard of care.

Efficacy Measures

The range of motion in the study knee was examined by the investigatoror nominee for subjects with limited range of motion due to pain andinflammation at pre-dose, 24 and 72 hours post-dose and the global painassessment (pain numerical rating scale) was performed at pre-dose, 6,24 and 72 hours post-dose and Western Ontario McMaster UniversityOsteoarthritis (WOMAC) Index 3.1 was completed by subjects at pre-dose,24 and 72 hours post-dose.

The pain numerical rating scale (NRS) in the study knee was completed atpre-dose, 6 hours post-dose on Day 1, 24 hours post-dose on Day 2 and 72hours post-dose on Day 4. The pain numerical rating scale was completedprior to WOMAC if the pain scale and WOMAC occur at the same time. Thepain NRS is a numerical rating of 0-10, with 0 being no pain, 5 beingmoderate pain and 10 being worst possible pain.

The range of motion (degrees of flexion and extension) in the study kneewas examined by the investigator or nominee for subjects with limitedrange of motion due to pain and inflammation not by osteophyte growth,at baseline, 24 hours post-dose on Day 2 and 72 hours post-dose on Day4.

WOMAC Index 3.1 was completed by subjects within 1 hour prior toinjection, 24 hours post-dose on Day 2 and 72 hours post-dose on Day 4.The WOMAC Index assesses joint function and pain in subjects with hip orknee osteoarthritis. It measures 24 parameters (questions) with eachparameter (question) being rated by the subject on a scale of 1-10.There are three subscores: pain (5 questions); stiffness (2 questions);and function (17 questions).

The data were analyzed using the Student's t-test: mean (SD) differencebetween treatment groups A and C for the following:

-   -   Mean change in pain NRS at 6 hours, 24 hours and 72 hours (FIG.        1);    -   Mean change in WOMAC pain subscores at 24 hours and 72 hours        (FIGS. 3A and 3B);    -   Mean change in WOMAC function subscores at 24 hours and 72 hours        (FIGS. 5A and 5B); and    -   Mean change in WOMAC stiffness subscores at 24 hours and 72        hours (FIGS. 4A and 4B).

Also, repeated measures ANOVA were calculated: mean (SD) differencebetween treatment groups A and C for mean change in pain NRS with time(FIG. 2).

Results

Results are presented in FIGS. 1-6, Tables 2 and 3 and are summarizedhere:

-   -   A trend in improvement in pain NRS with the <5000 MW Fraction        (Ampion™) was observed. This trend increased with time versus        steroids and demonstrates that the effects of the <5000 MW        Fraction are long lasting.    -   WOMAC—no differences were observed in the three subscores        analyzed.    -   The <5000 MW Fraction does not increase adverse events.    -   Data on rescue medications and range of motion were only        collected in a small percentage of patients and were not        analyzed.    -   Steroid Potential Time course is shown in FIG. 6. The onset of        steroids efficacy is rapid: 12-24 hours maximal effect is        reached in 1 week and lasts 4 weeks (Ann Rheum Dis 2004;        63:478-482). Short term pain increases from baseline through day        3 and decreases afterwards (Am J Orthop. 2007 March; 36(3):        128-31).

TABLE 2 Baseline Efficacy Variables, mean (SD) Efficacy Ampion ™ +Steriod + Steroid + T-test Variable lidocaine lidocane p value Pain NRS4.29 (2.28) 3.61 (1.69) 0.32 WOMAC Pain 4.09 (1.95) 3.99 (2.07) 0.91WOMAC Stiffness 3.86 (1.78) 3.93 (2.05) 0.54 WOMAC Function 5.15 (2.10)4.69 (2.26) 0.89 Ampion + Steroid + lidocane: n = 20, safety population;n = 17, efficacy analysis population Steroid + lidocane: n = 20, safetypopulation; n = 18, efficacy analysis population

TABLE 3 Mean Change in Pain NRS, mean (SD) Ampion ™ + Steriod +Steroid + T-test Time lidocaine lidocane p value  6 hours −2.31 (2.36)−2.06 (1.86) 0.73 24 hours −2.12 (1.58) −1.61 (2.15) 0.43 72 hours −2.50(1.83) −1.82 (1.74) 0.30

Example 2

A clinical trial was performed to investigate the effect ofintra-articular knee injection of the <5000 MW Fraction (also referredto herein as “Ampion™”) for improving joint function and reducing thepain of osteoarthritis of the knee in adults with symptomatic primaryknee osteoarthritis. A randomized, placebo-controlled, double-blind,parallel study with 43 evaluable subjects was chosen as the appropriatedesign to estimate the treatment effect and safety of the <5000 MWFraction when it was injected into the study knee.

Study Drug

2 arms; each subject received a single 4 ml injection in one knee withone of either Amipon™ or saline.

Study Population

The study population was 43 patient, male or female 40-83 years old(average 63.0, standard deviation (SD) 9.6) 28 were male and 15 werefemale. All subjects were Caucasian. The subjects' height ranged from162 to 192 cm (average 175.3, SD 8.1) with weight at screening rangingfrom 56 to 117 kg (average 88.8, SD 13.89). The subjects were fullyambulatory, with symptomatic primary knee osteoarthritis for more than 6months prior to screening with Kellgren Lawrence Grade II or III(indicating mild or moderate osteoarthritis). Grade II for 6 subjectsand Grade III for 36 subjects. One subject did have Grade IV. If bothknees of a subject were osteroarthritic, one knee was selected for studywhile the other knee received standard of care.

Exclusion Criteria:

The following is the exclusion criteria for the study population:

-   -   1. Unfit as a result of medical review and screening        investigation    -   2. A history of allergic reactions to albumin    -   3. A history of allergic reactions to excipients in 5% human        albumin    -   4. Any intra-articular or local periarticular injection,        injection or surgery to the index knee (previous 6 months)    -   5. Operative arthroscopy (previous 3 months)    -   6. Surgical procedure to the index knee other than arthroscopy        (previous 12 months)    -   7. Any investigational knee products (previous 12 months)    -   8. Kellgren Lawrence Grade I or IV (doubtful or severe)        osteoarthritis of the knee.    -   9. Inflammatory or crystal arthropathies, acute fractures,        severe loss of bone density, bone necrosis.    -   10. Isolated patella-femoral syndrome or chondromalacia.    -   11. Any other disease or condition interfering with the free use        and evaluation of the index knee    -   12. Major injury to the index (previous 12 months)    -   13. Severe hip osteoarthritis ipsilateral to the index knee.    -   14. Any pain that could interfere with the assessment of index        knee pain    -   15. Any pharmacological or non-pharmacological treatment started        or changed (previous 4 weeks)    -   16. Use of a. any topical treatment (previous 48 h) b. All        analgesics and NSAIDs except paracetamol (previous 48 h), c.        Anticoagulant therapy (previous 48 h) d. Any systemic steroid        treatments (previous 14 days), e. All immunosuppressives within        a period of 5 times the drug's half life prior to        randomization, f. corticosteroids >10 mg prednisolone equivalent        per day (previous 30 days), g. Any albumin treatment (previous 3        months)    -   17. Female subjects who are pregnant or lactating.    -   18. Female subjects of childbearing potential who have a        positive pregnancy test on Day 1 prior to treatment.

Study Assessment

The study consisted of a three week screening period and an 84 day studyparticipation period. Follow-up assessments were performed at 6 hours,24 hours and 72 hours post injection. Subjects were contact by telephoneat Day 8, Day 30 and Day 84 to evaluate overall pain and mobility and tomonitor adverse events. The subjects were offered the option ofintra-articular betamethasone injection to the investigative knee forpain relief after Day 8, if deemed necessary following an assessment bythe investigator.

Primary Outcome

The pain numerical rating scale (NRS) in the study knee was completed atpre-dose (pre-injection baseline), 6 hours post-dose on Day 1, 24 hourspost-dose on Day 2, 72 hours post-dose on Day 4, and at Day 8, Day 30and Day 84 post-dose (EOS or End-of-study). The pain NRS is a numericalrating of 0-10, with 0 being no pain, 5 being moderate pain and 10 beingworst possible pain.

Safety Endpoints

The safety endpoints of the study were incidence of adverse events,vital signs at pre-dose and study Day 4, twelve lead ECG readings atscreening and 24 hours post-dose, and clinical blood safety tests(biochemical and hematology) assessed at screening and 24 hourspost-dose.

Secondary Endpoints

The secondary endpoints of the study were percent responders at Day 30and Day 84, defined as an improvement in pain NRS of 2 or more points,the change from pre-injection baseline in WOMAC Osteoarthritis Index 3.1(complete scale, pain subscore, stiffness subscore and functionsubscore) at 24 and 72 hours after intra-articular injection, the changefrom pre-injection baseline for requirement for rescue medications(paracetamol) to 24 hours and 72 hours after intra-articular injectionand changes over time in mobility at Day 8, Day 30 and Day 84 post-dosecompared with pre-dose and the immediate post-dose period.

Intent to Treat (ITT) and Safety Population

Study participants who were randomized and received at least one dose ofthe study medication. ITT refers to subjects that metinclusion/exclusion criteria.

Per Protocol Population

Study participants in the ITT set whose pre-dose pain score did notviolate inclusion/exclusion criteria.

Efficacy Population

Study participants in the pre-protocol population who did not receiverescue medication between 8 and 30 days.

Statistical Analyses

Primary: Analysis of covariance (ANCOVA) model to examine the mean (SD)difference between treatment groups for mean change in pain at Day 30and Day 84 (EOS), adjusted for baseline pain NRS.

Additional: X² test for differences in percent responders.Cochran-armitage trend test for differences in clinically significantimprovements. Student's t-test: mean (SD) difference in pain NRS at 30days.

Safety Analysis:

Adverse events and serious adverse events were listed by subject.Summaries were presented by treatment of adverse events classified byMedDRA System Organ Class and Preferred Term, for overall incidence andby severity and relationship to study medication. Incidence oftreatment-emergent adverse events were compared between treatmentgroups. All clinical safety and tolerability data was listed for eachsubject and summarized by treatment. Vital signs and ECG parameters weretabulated and summarized by treatment. Laboratory values were listed,along with comments as to clinical significance for values outside thelaboratory's normal ranges. Changes from screening were assessed forclinical significance.

Results:

TABLE 4 Population Analysis Set Study Size(n) Ampion ™ (n) Saline (n)Safety Set 43 22 21 ITT Set 43 22 21 Per-protocol Set^(a) 41 20 21Efficacy 32 17 15 evaluable Set^(b) ^(a)2 subjects in the Ampion ™ grouphad baseline pain NRS < 4 points ^(b)5 subjects in the Ampion ™ groupand 6 subjects in the saline group required rescue medication

Use of Rescue Medications

Betamethasone injection: there was no apparent difference between theuse of betamethasone injections between subjects who received Ampion™ (5of 22 subjects, 23%) compared with subjects who received saline (6 of 21subjects, 29%).

Rescue medications (paracetamol): rescue medication for pain relief inthe study knee within 24 hours of injection occurred in a similar numberof subjects receiving Ampion™ (6 of 22 subjects) compared with subjectsreceiving saline (6 of 21 subjects), with similar mean doses ofparacetamol used in each of the treatment groups.

Efficacy Results:

TABLE 5 Pain NRS by treatment, mean (SD) pre-protocol population: 24 h72 h Day 8 Day 30 Day 84 Pre- 6 h Post Post Post Post Post Treatmentdose Post dose dose dose dose dose dose Ampion ™ 4.70 2.00 3.20 2.602.90 2.90 3.21 (0.7) (1.3) (1.5) (2.1) (2.1) (1.8) (1.8) Saline 5.292.67 3.00 2.86 3.33 3.86 4.81 (1.4) (1.9) (1.7) (2.1) (1.9) (2.2) (2.3)

TABLE 6 Least Squares (LS) Mean Change in Pain NRS: per-protocolpopulation 6 h 24 h 72 h Day 8 Day 30 Day 84 Treatment Post dose Postdose Post dose Post dose Post dose Post dose D (Ampion ™) −3.06 −1.69−2.31 −2.00 −2.16 −1.60 E (Saline) −2.28 −2.11 −2.22 −1.76 −1.09 −0.36 Pvalue 0.15 0.42 0.89 0.71 0.12 0.07 Scale: −10 = largest possibleimprovement in pain from baseline, 10 = smallest possible improvement(largest increase) in pain from baseline. Day 1: 6 hours post-dose*adjusted for baseline pain NRS

TABLE 7 LS Mean Change in Pain NRS: efficacy evaluable population 6 h 24h 72 h Day 8 Day 30 Day 84 Treatment Post dose Post dose Post dose Postdose Post dose Post dose D (Ampion ™) −2.91 −1.99 −2.94 −2.45 −2.29−2.22 E (Saline) −2.62 −2.61 −2.79 −2.22 −1.17 −0.46 P value 0.62 0.190.79 0.71 0.19 0.04 Scale: −10 = largest possible improvement in painfrom baseline, 10 = smallest possible improvement (largest increase) inpain from baseline. Day 1: 6 hours post-dose *adjusted for baseline painNRSPercent responders at Day 84 (EOS): per-protocol population (see Table8)Responder: decrease in Day 84 pain NRS of −2 to −10 points (with −10being the largest possible improvement in pain).Non-responder: decrease in pain at Day 84 of −1 to 10 (with 10 being thelargest possible increase in pain).

TABLE 8 Trends in pain at 30 days from baseline, by treatment group:Treatment Non-Responder Responder P value Ampion ™ 47.4% 52.6% 0.06Saline 76.2% 23.8%Percent responders at Day 84 (EOS): efficacy evaluable population (seeTable 9)Responder: decrease in Day 84 pain NRS of −2 to −10 points (with −10being the largest possible improvement in pain).Non-responder: decrease in pain at Day 84 of −1 to 10 points (with 10being the largest possible increase in pain).

TABLE 9 Trends in pain at 30 days from baseline, by treatment group:Treatment Non-Responder Responder P value Ampion ™ 35.7% 64.3% 0.10Saline 66.7% 33.3%

Summary of Findings: Efficacy:

Overall pain (as assessed by the pain numerical rating score) and WOMACscores were reduced post-dose for each of the treatment groups for theduration of the study (p<0.05), except placebo at Day 84. In addition,there was a trend in a significant difference between changes frombaseline at Day 30 and at Day 84 for subjects who received Ampion™compared to subjects who received saline placebo (Day 30: p=0.12; Day84: p=0.07). This trend became statistically significant in subjects whodid not receive rescue medication (p=0.04). There was a trend towards ahigher percentage of responders at the end of the study (Day 84) forsubjects receiving Ampion™ vs. Placebo (p=0.06). Use of paracetamolrescue medication up to 72 hours post-dose was highest in the TreatmentE group (saline). See FIG. 7.

Adverse Events (AEs)

Treatment-emergent AEs were reported for 20 of the 43 subjects (47%)following dose administration, with a total of 27 AEs. Commonlyoccurring AEs were headache and joint swelling and stiffness in theknee. Most subjects reported AEs classified as mild only (16 of 43subjects, 37%). Only 4 subjects (9%) reported AEs of moderate severity:

-   -   Ampion™: Joint injury and hypertension    -   Saline: Back pain and vessel puncture site haematoma

There were no apparent differences in the incidence of moderate AEsbetween subjects who received Ampion™ (2 subjects, 9%) compared withsubjects who received saline (2 subjects, 10%). These AEs were alldeemed to be probably not or definitely not related to study drug.

There were no AEs classified as severe.

AEs deemed to be related to study drug administration (possibly) werereported in 3 of 43 subjects (7%). There were no apparent differences inthe incidence of related AEs between subjects who received Ampion™ (1subject, 5%) compared with subjects who received saline (2 subjects,10%):

-   -   a. Headache of mild severity which commenced 5 minutes after        treatment administration and resolved 1.8 hours later (Ampion™)    -   b. Headache of mild severity which commenced 5 hours after        treatment administration and resolved 0.5 hours later (saline)    -   c. Joint swelling of right knee (study knee) of mild severity        which commenced 2.4 days after treatment administration and        resolved 21 hours later (saline)

Overall, a higher proportion of treatment-emergent AEs were reported insubjects who received saline (12 subjects, 57%) compared with subjectswho received Ampion™ (8 subjects, 36%). AEs deemed to be related tostudy drug administration (possibly) were reported in 3 of 43 subjects(7%) and included headache and joint swelling of the knee. There were nodeaths or other serious AEs. There were no clear differences in safetyas assessed by biochemistry clinical laboratory tests, vital signs, andECG assessments between treatments.

Conclusions of Study:

Pain (as assessed by the pain numerical rating score) and WOMAC scoreswere reduced post-dose for each of the treatment groups for the durationof the study, except placebo at Day 84, with no significant differencesbetween treatment groups. Despite a higher baseline pain NRS for thesaline group compared to the Ampion™ group, there was a trend towards along-term effect of study drug, with a higher percentage of subjects whoresponded at Day 84 for Ampion™ compared to saline. In subjectsreceiving Ampion™, overall pain was reduced post-dose for the durationof the study, whereas subjects receiving saline did not have a reductionin pain post-dose at Day 84. Use of paracetamol rescue medication up to72 hours post-dose was highest in the Treatment E group (saline).Ampion™ was considered safe and well tolerated at the dose used in thestudy.

Example 3

This study demonstrates that DA-DKP and N-Acetyl Tryptophan (NAT) of the<5 kDa fraction of 5% HSA binds to collagen. In particular, at equimolarconcentration, NAT binds more to oxidized collagen IV than DA-DKP.

Methods

Collagen source: 1 mg/ml Collagen IV form human placenta in 0.25% aceticacid (Sigma™). The collagen was oxidixed with 500 μM H2O2 in 1×PBS for 1hour at 37° C. The oxidized collagen IV (final=0.2 mg/ml) was thenincubated with 2 μM DA-DKP or 2 NAT in PBS for 1 hour at 37° C. At theend of the incubation, the <5 kDa fraction was isolated by a VIVASPIN® 2microcentrifugal filter having a molecular weight cutoff of 5,000. The<5 kDa fraction was analyzed for DA-DKP and/or NAT content using aliquid chromatograph-mass spectrometery (LCMS) method for DA-DKPanalysis.

The results of the analyses are shown in Table 10.

TABLE 10 Potential Binding of NAT and DA-DKP to H2O2 treated HumanCollagen Average % Standard Sample decrease Deviation 2 μM DA-DKP +Collagen IV 26.0% 4.2% 2 μM DA-DKP + Collagen IV w/2 μM NAT 25.6% 5.7% 2μM NAT + Collagen IV 48.9% 1.3% 2 μM NAT + Collagen IV w/2 μM DA-DKP28.7% 9.4%

The results provided in Table 10 demonstrate that DA-DKP and NAT bind tooxidized forms of collagen and hence block the oxidized sites fromtriggering immune cell activation.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing exemplary claims.

1.-27. (canceled)
 28. A method of treating joint pain in a humancomprising administering to a joint of the human selected from the groupconsisting of a shoulder joint, a hand joint and a spinal joint, anintra-articular injection of an effective amount of a pharmaceuticalcomposition comprising DA-DKP prepared by removing albumin from asolution of a human serum albumin composition.
 29. The method of claim28 wherein the composition is administered at a frequency selected fromthe group consisting of: a. no more frequently than once every sixmonths, b. no more frequently than once every 5 months, c. no morefrequently than once every 4 months, d. no more frequently than onceevery 3 months, and e. no more frequently than once every 2 months. 30.The method of claim 28, wherein the composition administered byintra-articular injection is a composition having a concentration ofDA-DKP from 50 μM to 350 μM.
 31. The method of claim 28, wherein thecomposition further comprises N-acetyl-tryptophan (NAT), caprylic acid,caprylate or combinations thereof.
 32. The method of claim 31, whereinthe composition is a composition having a concentration of NAT, caprylicacid, caprylate or combinations thereof from 4 mM to 20 mM.
 33. Themethod of claim 28, wherein the step of removing the albumin comprisestreating the human serum albumin composition by a separation methodselected from the group consisting of ultrafiltration, sucrose gradientcentrifugation, chromatography, salt precipitation, and sonication. 34.The method of claim 33, wherein the step of removing comprises passingthe human serum albumin composition over an ultrafiltration membranewith a molecular weight cut off that retains the albumin, and whereinthe resulting filtrate comprises DA-DKP.
 35. The method of claim 34,wherein the ultrafiltration membrane has a molecular weight cutoffselected from the group consisting of less than 50 kDa, less than 40kDa, less than 30 kDa, less than 20 kDa, less than 10 kDa, less than 5kDa and less than 3 kDa.
 36. The method of claim 28, wherein thepharmaceutical composition further comprises a second drug selected fromthe group consisting of an analgesic, an anti-inflammatory drug, andcombinations thereof.
 37. A method of treating joint pain in a humancomprising administering to a joint of the human selected from the groupconsisting of a shoulder joint, a hand joint and a spinal joint, anintra-articular injection of an effective amount of a pharmaceuticalcomposition comprising DA-DKP at a concentration of from 50 μM to 350 μMprepared by passing the human serum albumin composition over anultrafiltration membrane with a molecular weight cut off that retainsthe albumin, and wherein the resulting filtrate comprises DA-DKP. 38.The method of claim 37, wherein the composition is a composition havinga concentration of NAT, caprylic acid, caprylate or combinations thereoffrom 4 mM to 20 mM.
 39. The method of claim 37, wherein theultrafiltration membrane has a molecular weight cutoff selected from thegroup consisting of less than 50 kDa, less than 40 kDa, less than 30kDa, less than 20 kDa, less than 10 kDa, less than 5 kDa and less than 3kDa.
 40. The method of claim 37, wherein the ultrafiltration membranehas a molecular weight cutoff of less than 5 kDa.
 41. A method oftreating osteoarthritis in a human comprising administering to a jointof the human selected from the group consisting of a shoulder joint, ahand joint and a spinal joint, an effective amount of a pharmaceuticalcomposition comprising DA-DKP prepared by removing albumin from asolution of a human serum albumin composition.
 42. The method of claim41, wherein the composition is a composition having a concentration ofNAT, caprylic acid, caprylate or combinations thereof from 4 mM to 20mM.
 43. The method of claim 41, wherein the ultrafiltration membrane hasa molecular weight cutoff selected from the group consisting of lessthan 50 kDa, less than 40 kDa, less than 30 kDa, less than 20 kDa, lessthan 10 kDa, less than 5 kDa and less than 3 kDa.
 44. The method ofclaim 41, wherein the ultrafiltration membrane has a molecular weightcutoff of less than 5 kDa.
 45. The method of claim 41, wherein thepharmaceutical injection is by intra-articular injection to the jointselected from the group consisting of a shoulder joint, a hand joint anda spinal joint of the human.